Amylolytic Enzyme Variants

ABSTRACT

The inventors have discovered some striking, and not previously predicted structural similarities and differences between the structure of Novamyl and the reported structures of CGTases, and based on this they have constructed variants of maltogenic alpha-amylase having CGTase activity and variants of CGTase having maltogenic alpha-amylase activity. Further, on the basis of sequence homology between Novamyl® and CGTases, the inventors have constructed hybrid enzymes with one or more improvements to specific properties of the parent enzymes, using recombinant DNA methodology.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/453,828 filed May29, 2003 which is a divisional of U.S. Ser. No. 10/1234,266, filed onSep. 4, 2002, which is a divisional of U.S. Ser. No. 09/645,707, filedon Aug. 24, 2000 (now U.S. Pat. No. 6,482,622), which is a continuationof PCT/DK99/00087, filed on Feb. 26, 1999, and claims priority under 35U.S.C. 119 of Danish application nos. PA 1998 00269 and PA 1998 00273,both filed on Feb. 27, 1998, and U.S. provisional application Nos.60/077,509 and 60/077,795, filed on Mar. 11, 1998 and Mar. 12, 1998,respectively, the contents of which are fully incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to methods of converting a maltogenicalpha-amylase into a cyclodextrin glucanotransferase (CGTase) or viceversa or creating hybrids of the two. The invention also relates to thevariants made by the methods.

BACKGROUND OF THE INVENTION

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) and maltogenicalpha-amylase (EC 3.2.1.133) are two classes of glycosylases thatdegrade starch by hydrolysis of the α-(1,4)-glycosidic bonds, but theinitial products are predominantly cyclic for CGTases and linear for themaltogenic alpha-amylase.

Cyclomaltodextrin glucanotransferase (E.C. 2.4.1.19), also designatedcyclodextrin glucanotransferase or cyclodextrin glycosyltransferase,abbreviated herein as CGTase, catalyses the conversion of starch andsimilar substrates into cyclomaltodextrins via an intramoleculartransglycosylation reaction, thereby forming cyclomaltodextrins (or CD)of various sizes. Commercially most important are cyclodextrins of 6, 7and 8 glucose units, termed α-, β- and γ-cyclodextrins, respectively.

CGTases are widely distributed and from several different bacterialsources, including Bacillus, Brevibacterium, Clostridium,Corynebacterium, Klebsiella, Micrococcus, Thermoanaerobacter andThermoanaerobacterium have been extensively described in the literature.A CGTase produced by Thermoanaerobacter sp. has been reported in NormanB E, Jørgensen S T; Denpun Kaqaku 1992 39 99-106, and WO 89/03421, andthe amino acid sequence has been disclosed in WO 96/33267. The sequenceof CGTases from Thermoanaerobacterium thermosulfurigenes and fromBacillus circulansis available on the Internet (SCOP or PDF home pages)as pdf file 1CIU, and the sequence of a CGTase from B. circulans isavailable as pdf file 1CDG.

Tachibana, Y., Journal of Fermentation and Bioengineering, 83 (6),540-548 (1997) describes the cloning and expression of a CGTase.Variants of CGTases have been described by Kim, Y. H., Biochemistry andMolecular Biology International, 41 (2), 227-234 (1997); Sin K-A,Journal of Biotechnology, 32 (3), 283-288 (1994); D Penning a,Biochemistry, 34 (10), 3368-3376 (1995); and WO 96/33267.

Maltogenic alpha-amylase (glucan 1,4-a-maltohydrolase, E.C. 3.2.1.133)is able to hydrolyze amylose and amylopectin to maltose in thealpha-configuration, and is also able to hydrolyze maltotriose as wellas cyclodextrin.

A maltogenic alpha-amylase from Bacillus (EP 120 693) is commerciallyavailable under the trade name Novamyl® (product of Novo Nordisk A/S,Denmark) and is widely used in the baking industry as an anti-stalingagent due to its ability to reduce retrogradation of starch (WO91/04669).

The maltogenic alpha-amylase Novamyl® shares several characteristicswith cyclodextrin glucanotransferases (CGTases), including sequencehomology (Henrissat B, Bairoch A; Biochem. J., 316, 695-696 (1996)) andformation of transglycosylation products (Christophersen, C., et al.,1997, Starch, vol. 50, No. 1, 39-45).

BRIEF DESCRIPTION OF THE INVENTION

The inventors have discovered some striking, and not previouslypredicted structural similarities and differences between the structureof Novamyl and the reported structures of CGTases, and based on thisthey have constructed variants of maltogenic alpha-amylase having CGTaseactivity and variants of CGTase having maltogenic alpha-amylaseactivity. Further, on the basis of sequence homology between Novamyl®and CGTases, the inventors have constructed hybrid enzymes with one ormore improvements to specific properties of the parent enzymes, usingrecombinant DNA methodology.

Accordingly, the present invention provides a polypeptide which:

a) has at least 70% identity to amino acids 1-686 of SEQ ID NO: 1;

b) comprises an amino acid modification which is an insertion,substitution or deletion compared to SEQ ID NO: 1 in a regioncorresponding to amino acids 40-43, 78-85, 136-139, 173-180, 188-195 or259-268; and

c) has the ability to form cyclodextrin when acting on starch.

The invention also provides a polypeptide which:

a) has an amino acid sequence having at least 70% identity to a parentcyclodextrin glucanotransferase (CGTase);

b) comprises an amino acid modification which is an insertion,substitution or deletion compared to the parent CGTase in a regioncorresponding to amino acids 40-43, 78-85, 136-139, 173-180, 188-195 or259-268 of SEQ ID NO: 1; and

c) has the ability to form linear oligosaccharides when acting onstarch.

Further, the invention provides a method for constructing a maltogenicalpha-amylase, comprising:

a) recombining DNA encoding a cyclodextrin glucanotransferase (CGTase)and DNA encoding a maltogenic alpha-amylase;

b) using the recombinant DNA to express a polypeptide; and

c) testing the polypeptide to select a polypeptide having the ability toform linear oligosaccharides when acting on starch.

Finally, the invention provides a method of selecting DNA encodingmaltogenic alpha-amylase in a DNA pool, comprising:

a) amplifying DNA encoding maltogenic alpha-amylase by a polymerasechain reaction (PCR) using primers encoding a partial amino acidsequence of amino acids 1-686 of SEQ ID NO: 1, preferably comprising atleast 5 amino acid residues, preferably comprising one or more ofpositions 188-196, more preferably comprising positions 190-194,

b) cloning and expressing the amplified DNA, and

c) screening for maltogenic alpha-amylase activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows plasmid pCA31, described in Example 1.

FIG. 2 is a diagram showing the shuffling of Novamyl with CGTases, asdescribed in Example 3.

FIG. 3 is a diagram showing the selection of clones with desiredfeatures by PCR, as described in Examples 2 and 3.

FIG. 4 a-4 b shows an alignment of the amino acid sequence of Novamyl(1-686 of SEQ ID NO: 1) with the sequence of 3 CGTases as describedbelow.

DETAILED DESCRIPTION OF THE INVENTION Maltogenic Alpha-Amylase

The parent maltogenic alpha-amylase used in the invention is an enzymeclassified in EC 3.2.1.133. The enzymatic activity does not require anon-reducing end on the substrate and the primary enzymatic activityresults in the degradation of amylopectin and amylose to maltose andlonger maltodextrins. It is able to hydrolyze amylose and amylopectin tomaltose in the alpha-configuration, and is also able to hydrolyzemaltotriose as well as cyclodextrin.

A particularly preferred maltogenic alpha-amylase is the amylase clonedfrom Bacillus as described in EP 120 693 (hereinafter referred to asNovamyl). Novamyl has the amino acid sequence set forth in amino acids1-686 of SEQ ID NO: 1. Novamyl is encoded in the gene harbored in theBacillus strain NCIB 11837 which has the nucleic acid sequence set forthin SEQ ID NO:1.

CGTase

The parent CGTase used in the invention is an enzyme classified in EC2.4.1.19. It may be from any source, e.g. bacterial sources, includingBacillus, Brevibacterium, Clostridium, Corynebacterium, Klebsiella,Micrococcus, Thermoanaerobacter and Thermoanaerobacterium.

The CGTase preferably has one or more of the following characteristics:

i) an amino acid sequence having at least 50% identity to amino acids1-686 of SEQ ID NO: 1, preferably at least 60%;

ii) being encoded by a DNA sequence which hybridizes at conditionsdescribed below to the DNA sequence set forth in SEQ ID NO:1 or to theDNA sequence encoding Novamyl harbored in the Bacillus strain NCIB11837; and

iii) a catalytic binding site comprising amino acid residuescorresponding to D228, E256 and D329 as shown in the amino acid sequenceset forth in amino acids 1-686 of SEQ ID NO: 1.

Variants of CGTase

The CGTase variant of this invention has the ability to form linearoligosaccharides when acting on starch. The starch hydrolysis and theanalysis of initial reaction products may be carried out as described inan Example.

The CGTase variant has a modification of at least one amino acid residuein a region corresponding to residues 40-43, 78-85, 136-139, 173-180,189-195 or 259-268 of SEQ ID NO: 1. Each modification may be aninsertion, a deletion or a substitution, of one or more amino acidresidues in the region indicated. The modification of the parent CGTaseis preferably such that the resulting modified amino acid or amino acidsequence more closely resembles the corresponding amino acid orstructural region in Novamyl. Thus, the modification may be an insertionof or a substitution with an amino acid present at the correspondingposition of Novamyl, or a deletion of an amino acid not present at thecorresponding position of Novamyl.

The CGTase variant may particularly comprise an insertion into aposition corresponding to the region D190-F194 of Novamyl (amino acidsequence shown in SEQ ID NO: 1). The insertion may comprise 3-7 aminoacids, particularly 4-6, e.g. 5 amino acids. The insertion may be DPAGF(SEQ ID NO:27) as found in Novamyl or an analogue thereof, e.g. with thefirst amino acid being negative, the last one being aromatic, and theones in between being preferably P, A or G. The variant may furthercomprise a substitution at the position corresponding to T189 of Novamylwith a neutral amino acid which is less bulky than F, Y or W. Otherexamples of insertions are DAGF (SEQ ID NO:28), DPGF (SEQ ID. NO:29),DPF, DPAAGF (SEQ ID NO:30), and DPAAGGF (SEQ ID NO:31).

Modifications in the region 78-85 preferably include deletion of 2-5amino acids, e.g. 3 or 4. Preferably, any aromatic amino acid in theregion 83-85 should be deleted or substituted with a non-aromatic.

Modifications in the region 259-268 preferably include deletion of 1-3amino acid, e.g. two. The region may be modified so as to correspond toNovamyl

The CGTase variant may comprise further modifications in other regions,e.g. regions corresponding to amino acids 37-39, 44-45, 135, 140-145,181-186, 269-273, or 377-383 of Novamyl.

Additional modifications of the amino acid sequence may be modeled on asecond CGTase, i.e. an insertion of or substitution with an amino acidfound at a given position in the second CGTase, or they may be madeclose to the substrate (less than 8 Å from the substrate, e.g. less than5 Å or less than 3 Å) as described in WO 96/33267.

The following are some examples of variants based on a parent CGTasefrom Thermoanaerobacter (using B. circulans numbering). Similar variantsmay be made from other CGTases.

L194F+*194aT+*194bD+*194cP+*194dA+*194eG+D196S

L87H+D89*+T91G+F91aY+G92*+G93*+S94*+L194F+*194aT+*194bD+*194cP+*194dA+*194eG+D196S

*194aT+*194bD+*194cP+*194dA+*194eG+D196S

L87H+D89*+T91G+F91aY+G92*+G93*+S94*+*194aT+*194bD+*194cP+*194dA+*194eG+D196S

Y260F+L261G+G262D+T263D+N264P+E265G+V266T+*266aA+*266bN+D267H+P268V

*194aT+*194bD+*194cP+*194dA+*194eG+D196S+Y260F+L261G+G262D+T263D+N264P+E265G+V266T+*266aA+*266bN+D267H+P268V

Variants of Novamyl

The Novamyl variant of this invention has the ability to formcyclodextrin when acting on starch. The starch hydrolysis and theanalysis of reaction products may be carried out as described in anExample.

The Novamyl variant has a modification of at least one amino acidresidue in the same regions described above for CGTase variants.However, the modifications are preferably in the opposite direction,i.e. such that the resulting modified amino acid or amino acid sequencemore closely resembles the corresponding amino acid or structural regionof a CGTase. Thus, the modification may be an insertion of or asubstitution with an amino acid present at the corresponding position ofa CGTase, or a deletion of an amino acid not present at thecorresponding position of a CGTase.

Preferred modifications include a deletion in the region 190-195,preferably the deletion Δ (191-195) and/or a substitution of amino acid188 and/or 189, preferably F188L and/or Y189Y.

Amino Acid Identity

For purposes of the present invention, the degree of identity may besuitably determined according to the method described in Needleman, S.B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-45,with the following settings for polypeptide sequence comparison: GAPcreation penalty of 3.0 and GAP extension penalty of 0.1. Thedetermination may be done by means of a computer program known such asGAP provided in the GCG program package (Program Manual for theWisconsin Package, Version 8, August 1994, Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711).

The variants of the invention have an amino acid identity with theparent enzyme (Novamyl or CGTase) of at least 70%, preferably at least80%, e.g. at least 90%, particularly at least 95% or at least 98%.

Hybridization

The hybridization referred to above indicates that the analogous DNAsequence hybridizes to the nucleotide probe corresponding to the proteinencoding part of the nucleic sequence shown in SEQ ID NO:1, under atleast low stringency conditions as described in detail below.

Suitable experimental conditions for determining hybridization at lowstringency between a nucleotide probe and a homologous DNA or RNAsequence involves presoaking of the filter containing the DNA fragmentsor RNA to hybridize in 5×SSC (sodium chloride/sodium citrate, Sambrook,J., Fritsch, E. J., and Maniatis, T. (1989) Molecular cloning: alaboratory manual, Cold Spring Harbor Laboratory Press, New York) for 10min, and prehybridization of the filter in a solution of 5×SSC,5×Denhardt's solution (Sambrook, et al., op.cit.), 0.5% SDS and 100μg/ml of denatured sonicated salmon sperm DNA (Sambrook, et al.,op.cit.), followed by hybridization in the same solution containing arandom-rimed (Feinberg, A P. and Vogelstein, B. (1983) Anal. Biochem.132:6-13), ³²P-dCTP-labeled (specific activity >1×10⁹ cpm/μg) probe for12 hours at ca. 45° C. The filter is then washed twice for 30 minutes in2×SSC, 0.5% SDS at least 55° C. (low stringency), more preferably atleast 60° C. (medium stringency), more preferably at least 65° C.(medium/high stringency), more preferably at least 70° C. (highstringency), even more preferably at least 75° C. (very highstringency).

Molecules which hybridize to the oligonucleotide probe under theseconditions are detected by exposure to x-ray film.

Corresponding Amino Acids

Corresponding amino acids for the following 4 amino acid sequences areshown in the alignment in FIG. 4 a-4 b which is based on thethree-dimensional structure of the sequences.

1) Novamyl (amino acids 1-686 of SEQ ID NO: 1)

2) CGTase from Thermoanaerobacterium thermosulfurigenes (pdf file 1CIU)

3) CGTase from Thermoanaerobacter, described in WO 96/33267

4) CGTase from Bacillus circulans (pdf file 1CDG)

Corresponding amino acid residues in other CGTases may be found byaligning with one of the sequences in FIG. 4 a-4 b by to the methoddescribed in Needleman (supra) using the same parameters, e.g. by meansof the GAP program (supra).

Nomenclature for Amino Acid Modifications

The nomenclature used herein for defining mutations is essentially asdescribed in WO 92/05249. Thus, F188L indicates a substitution of theamino acid F (Phe) in position 188 with the amino acid L (Leu). Δ(191-195) or Δ (191-195) indicates a deletion of amino acids inpositions 191-195. 192-A-193 indicates an insertion of A between aminoacids 192 and 193. *194aT indicates an insertion of T at the firstposition after 194. G92* indicates a deletion of G at position 92.

Recombination of CGTase and Maltogenic Alpha-Amylase

The present invention further relates to a method for constructing avariant enzyme comprising Novamyl and one or more parent CGTases,wherein said variant has at least one altered property relative toNovamyl and said parent CGTases, which method comprises:

i) generating DNA fragments encoding amino acid sequences obtainablefrom Novamyl and said parent CGTases;

ii) constructing a hybrid variant which contains amino acid sequencesgenerated in step i) by in vivo or in vitro DNA shuffling; and

iii) testing the resulting variant for said property.

The methods for generating DNA fragments referred to in step i) of themethod above are well known in the art and may include, for example,treatment of a DNA sequence encoding an amino acid sequence with arestriction enzyme, e.g., DNAse I.

The DNA shuffling referred to in step ii) in the method above may berecombination, either in vivo or in vitro, of nucleotide sequencefragment(s) between two or more polynucleotides resulting in outputpolynucleotides (i.e., polynucleotides having been subjected to ashuffling cycle) having a number of nucleotide fragments exchanged, incomparison to the input polynucleotides (i.e. starting pointpolynucleotides). Shuffling may be accomplished either in vitro or invivo by recombination within a cell by methods described in the art(cf., Crameri, et al, 1997, Nature Biotechnology Vol. 15:436-438).

In a preferred embodiment, at least one DNA fragment obtainable fromNovamyl in step i) of the method above encodes an amino acid sequence,which is determined to be of relevance for altering said property.

In a more preferred embodiment, a hybrid variant of a parent CGTase isobtained by the above method comprising a modification of at least oneamino acid residue in the group consisting of amino acid residuescorresponding to residues 37 to 45, residues 135 to 145, residues 173 to180, residues 189 to 196, residues 261 to 266, residues 327 to 330, andresidues 370 to 376 of SEQ ID NO: 1.

In another more preferred embodiment, a hybrid variant comprisingNovamyl and one or more parent CGTases is constructed by the abovemethod in which the amino acid sequence ofAsp190-Pro191-Ala192-Gly-193-Phe194 corresponding to the positions inthe amino acid sequence shown in SEQ ID NO: 1 is inserted into thecorresponding positions in said hybrid, wherein the correspondingpositions is determined on the basis of amino acid sequence alignment.

In another more preferred embodiment, a hybrid variant comprisingNovamyl and one or more parent CGTase is obtained by the above method inwhich the amino acid sequence ofAsp190-Pro191-Ala192-Gly193Phe194-Ser195 corresponding to the positionsin the amino acid sequence shown in SEQ ID NO: 1 is inserted into thecorresponding positions in said hybrid, wherein the correspondingpositions is determined on the basis of amino acid sequence alignment.

It is possible to use the unique active site loop to select hybridenzymes with maltogenic alpha-amylase activity from a library of randomrecombinants. Thus, a maltogenic alpha-amylase and a CGTase may berandomly recombined, e.g. by the DNA shuffling method of Crameri A, etal., op.cit. Those resulting mutants containing the Novamyl loop may beselected using PCR, e.g. as described above in the Examples.

The property to be altered may be substrate specificity, substratebinding, substrate cleavage pattern, specific activity of cleavage,transglycosylation, and relative activity of cyclization.

The DNA sequence encoding a parent CGTase to be used in the methods ofthe invention may be isolated from any cell or microorganism producingthe CGTase in question using methods known in the art.

Cloning a DNA Sequence Encoding a CGTaseCloning a DNA Sequence Encodingan a-AmylaseCloning a DNA Sequence Encoding an a-AmylaseCloning a DNASequence Encoding an a-AmylaseCloning a DNA Sequence Encoding ana-AmylaseCloning a DNA Sequence Encoding an a-AmylaseCloning a DNASequence Encoding an a-AmylaseCloning a DNA Sequence Encoding ana-AmylaseCloning a DNA Sequence Encoding an a-AmylaseCloning a DNASequence Encoding an a-Amylase

The DNA sequence encoding a parent CGTase may be isolated from any cellor microorganism producing the CGTase in question, using various methodswell known in the art, for example, from the Bacillus strain NCIB 11837.

First, a genomic DNA and/or cDNA library should be constructed usingchromosomal DNA or messenger RNA from the organism that produces theCGTase to be studied. Then, if the amino acid sequence of the CGTase isknown, homologous, labeled oligonucleotide probes may be synthesized andused to identify CGTase-encoding clones from a genomic library preparedfrom the organism in question. Alternatively, a labeled oligonucleotideprobe containing sequences homologous to a known CGTase gene could beused as a probe to identify CGTase-encoding clones, using hybridizationand washing conditions of lower stringency.

Another method for identifying CGTase-encoding clones involves insertingfragments of genomic DNA into an expression vector, such as a plasmid,transforming maltogenic alpha-amylase negative bacteria with theresulting genomic DNA library, and then plating the transformed bacteriaonto agar containing a substrate for maltogenic alpha-amylase, therebyallowing clones expressing maltogenic alpha-amylase activity to beidentified.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g. the phosphoroamiditemethod described by S. L. Beaucage and M. H. Caruthers (1981) or themethod described by Matthes et al. (1984). In the phosphoroamiditemethod, oligonucleotides are synthesized, e.g. in an automatic DNAsynthesizer, purified, annealed, ligated and cloned in appropriatevectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin,wherein the fragments correspond to various parts of the entire DNAsequence, in accordance with techniques well known in the art. The DNAsequence may also be prepared by polymerase chain reaction (PCR) usingspecific primers, for instance as described in U.S. Pat. No. 4,683,202or R. K. Saiki et al. (1988).

Random Mutagenesis

A general approach for modifying proteins and enzymes has been based onrandom mutagenesis, for instance, as disclosed in U.S. Pat. No.4,894,331 and WO 93/01285. For instance, the random mutagenesis may beperformed by use of a suitable physical or chemical mutagenizing agent,by use of a suitable oligonucleotide, or by subjecting the DNA sequenceto PCR generated mutagenesis. Furthermore, the random mutagenesis may beperformed by use of any combination of these mutagenizing agents. Themutagenizing agent may, e.g., be one which induces transitions,transversions, inversions, scrambling, deletions, and/or insertions.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues. When such agents are used, themutagenesis is typically performed by incubating the DNA sequenceencoding the parent enzyme to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions for themutagenesis to take place, and selecting for mutated DNA having thedesired properties.

When the mutagenesis is performed by the use of an oligonucleotide, theoligonucleotide may be doped or spiked with the three non-parentnucleotides during the synthesis of the oligonucleotide at the positionswhich are to be changed. The doping or spiking may be done so thatcodons for unwanted amino acids are avoided. The doped or spikedoligonucleotide can be incorporated into the DNA encoding the maltogenicalpha-amylase enzyme by any published technique, using e.g. PCR, LCR orany DNA polymerase and ligase as deemed appropriate.

Preferably, the doping is carried out using “constant random doping”, inwhich the percentage of wild-type and mutation in each position ispredefined. Furthermore, the doping may be directed toward a preferencefor the introduction of certain nucleotides, and thereby a preferencefor the introduction of one or more specific amino acid residues. Thedoping may be made, e.g., so as to allow for the introduction of 90%wild type and 10% mutations in each position. An additionalconsideration in the choice of a doping scheme is based on genetic aswell as protein-structural constraints. The doping scheme may be made byusing the DOPE program (cf., Tomandl, D. et al., 1997, Journal ofComputer-Aided Molecular Design 11:29-38; Jensen, L J, Andersen, K V,Svendsen, A, and Kretzschmar, T (1998) Nucleic Acids Research26:697-702) which, inter alia, ensures that introduction of stop codonsis avoided.

When PCR-generated mutagenesis is used, either a chemically treated ornon-treated gene encoding a parent CGTase enzyme is subjected to PCRunder conditions that increase the misincorporation of nucleotides(Deshler 1992; Leung et al., Technique, Vol. 1, 1989, pp. 11-15).

A mutator strain of E. coli (Fowler et al., Molec. Gen. Genet., 133,1974, pp. 179-191), S. cereviseae or any other microbial organism may beused for the random mutagenesis of the DNA encoding the CGTase by, e.g.,transforming a plasmid containing the parent CGTase into the mutatorstrain, growing the mutator strain with the plasmid and isolating themutated plasmid from the mutator strain. The mutated plasmid may besubsequently transformed into the expression organism.

The DNA sequence to be mutagenized may be conveniently present in agenomic or cDNA library prepared from an organism expressing the parentCGTase. Alternatively, the DNA sequence may be present on a suitablevector such as a plasmid or a bacteriophage, which as such may beincubated with or otherwise exposed to the mutagenising agent. The DNAto be mutagenized may also be present in a host cell either by beingintegrated in the genome of said cell or by being present on a vectorharbored in the cell. Finally, the DNA to be mutagenized may be inisolated form. It will be understood that the DNA sequence to besubjected to random mutagenesis is preferably a cDNA or a genomic DNAsequence.

In some cases it may be convenient to amplify the mutated DNA sequenceprior to expression or screening. Such amplification may be performed inaccordance with methods known in the art, the presently preferred methodbeing PCR-generated amplification using oligonucleotide primers preparedon the basis of the DNA or amino acid sequence of the parent enzyme.

Subsequent to the incubation with or exposure to the mutagenising agent,the mutated DNA is expressed by culturing a suitable host cell carryingthe DNA sequence under conditions allowing expression to take place. Thehost cell used for this purpose may be one which has been transformedwith the mutated DNA sequence, optionally present on a vector, or onewhich was carried the DNA sequence encoding the parent enzyme during themutagenesis treatment. Examples of suitable host cells are thefollowing: gram positive bacteria such as Bacillus subtilis, Bacilluslicheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium, Bacillus thuringiensis, Streptomyces lividans orStreptomyces murinus; and gram negative bacteria such as E. coli.

The mutated DNA sequence may further comprise a DNA sequence encodingfunctions permitting expression of the mutated DNA sequence.

DNA Shuffling

Alternative methods for rapid preparation of modified polypeptides maybe prepared using methods of in vivo or in vitro DNA shuffling whereinDNA shuffling is defined as recombination, either in vivo or in vitro,of nucleotide sequence fragment(s) between two or more polynucleotidesresulting in output polynucleotides (i.e., polynucleotides having beensubjected to a shuffling cycle) having a number of nucleotide fragmentsexchanged, in comparison to the input polynucleotides (i.e. startingpoint polynucleotides). Shuffling may be accomplished either in vitro orin vivo by recombination within a cell by methods described in the art.

For instance, Weber et al. (1983, Nucleic Acids Research, vol. 11,5661-5661) describe a method for modifying genes by in vivorecombination between two homologous genes, wherein recombinants wereidentified and isolated using a resistance marker.

Pompon et al., (1989, Gene 83:15-24) describe a method for shufflinggene domains of mammalian cytochrome P-450 by in vivo recombination ofpartially homologous sequences in Saccharomyces cereviseae bytransforming Saccharomyces cereviseae with a linearized plasmid withfilled-in ends, and a DNA fragment being partially homologous to theends of said plasmid.

In WO 97/07205 a method is described whereby polypeptide variants areprepared by shuffling different nucleotide sequences of homologous DNAsequences by in vivo recombination using plasmid DNA as template.

U.S. Pat. No. 5,093,257 (Genencor Int. Inc.) discloses a method forproducing hybrid polypeptides by in vivo recombination. Hybrid DNAsequences are produced by forming a circular vector comprising areplication sequence, a first DNA sequence encoding the amino-terminalportion of the hybrid polypeptide, a second DNA sequence encoding thecarboxy-terminal portion of said hybrid polypeptide. The circular vectoris transformed into a rec positive microorganism in which the circularvector is amplified. This results in recombination of said circularvector mediated by the naturally occurring recombination mechanism ofthe rec positive microorganism, which include prokaryotes such asBacillus and E. coli, and eukaryotes such as Saccharomyces cereviseae.

One method for the shuffling of homologous DNA sequences has beendescribed by Stemmer (Stemmer, (1994), Proc. Natl. Acad. Sci. USA, Vol.91, 10747-10751; Stemmer, (1994), Nature, vol. 370, 389-391; Crameri A,Dawes G, Rodriguez E Jr, Silver S, Stemmer W P C (1997) NatureBiotechnology Vol. 15, No. 5 pp. 436-438). The method concerns shufflinghomologous DNA sequences by using in vitro PCR techniques. Positiverecombinant genes containing shuffled DNA sequences are selected from aDNA library based on the improved function of the expressed proteins.

The above method is also described in WO 95/22625 in relation to amethod for shuffling homologous DNA sequences. An important step in themethod described in WO 95/22625 is to cleave the homologous templatedouble-stranded polynucleotide into random fragments of a desired sizefollowed by homologously reassembling of the fragments into full-lengthgenes.

Site-Directed Mutagenesis

Once a maltogenic alpha-amylase-encoding DNA sequence has been isolated,and desirable sites for mutation identified, mutations may be introducedusing synthetic oligonucleotides. These oligonucleotides containnucleotide sequences flanking the desired mutation sites; mutantnucleotides are inserted during oligonucleotide synthesis. In a specificmethod, a single-stranded gap of DNA, bridging the maltogenicalpha-amylase-encoding sequence, is created in a vector carrying themaltogenic alpha-amylase gene. Then the synthetic nucleotide, bearingthe desired mutation, is annealed to a homologous portion of thesingle-stranded DNA. The remaining gap is then filled in with DNApolymerase I (Klenow fragment) and the construct is ligated using T4ligase. A specific example of this method is described in Morinaga etal. (1984). U.S. Pat. No. 4,760,025 discloses the introduction ofoligonucleotides encoding multiple mutations by performing minoralterations of the cassette. However, an even greater variety ofmutations can be introduced at any one time by the Morinaga methodbecause a multitude of oligonucleotides, of various lengths, can beintroduced.

Another method of introducing mutations into a maltogenicalpha-amylase-encoding DNA sequences is described in Nelson and Long,Analytical Biochemistry 180, 1989, pp. 147-151. It involves a 3-stepgeneration of a PCR fragment containing the desired mutation introducedby using a chemically synthesized DNA strand as one of the primers inthe PCR reactions. From the PCR-generated fragment, a DNA fragmentcarrying the mutation may be isolated by cleavage with restrictionendonucleases and reinserted into an expression plasmid.

Localized Random Mutagenesis

The random mutagenesis may be advantageously localised to a part of theparent CGTase in question. This may, e.g., be advantageous when certainregions of the enzyme have been identified to be of particularimportance for a given property of the enzyme, and when modified areexpected to result in a variant having improved properties. Such regionsmay normally be identified when the tertiary structure of the parentenzyme has been elucidated and related to the function of the enzyme.

The localized, or region-specific, random mutagenesis is convenientlyperformed by use of PCR generated mutagenesis techniques as describedabove or any other suitable technique known in the art. Alternatively,the DNA sequence encoding the part of the DNA sequence to be modifiedmay be isolated, e.g., by insertion into a suitable vector, and saidpart may be subsequently subjected to mutagenesis by use of any of themutagenesis methods discussed above.

Expression of Maltogenic Alpha-Amylase Variants

The construction of the variant of interest is accomplished bycultivating a microorganism comprising a DNA sequence encoding thevariant under conditions which are conducive for producing the variant,and optionally subsequently recovering the variant from the resultingculture broth. This is described in detail further below.

According to the invention, a DNA sequence encoding the variant producedby methods described above, or by any alternative methods known in theart, can be expressed, in the form of a protein or polypeptide, using anexpression vector which typically includes control sequences encoding apromoter, operator, ribosome binding site, translation initiationsignal, and, optionally, a repressor gene or various activator genes.

The recombinant expression vector carrying the DNA sequence encoding anmaltogenic alpha-amylase variant of the invention may be any vectorwhich may conveniently be subjected to recombinant DNA procedures, andthe choice of vector will often depend on the host cell into which it isto be introduced. Thus, the vector may be an autonomously replicatingvector, i.e. a vector which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g. aplasmid, a bacteriophage or an extrachromosomal element, minichromosomeor an artificial chromosome. Alternatively, the vector may be one which,when introduced into a host cell, is integrated into the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated.

In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Examples of suitable promoters for directing thetranscription of the DNA sequence encoding a maltogenic alpha-amylasevariant of the invention, especially in a bacterial host, are thepromoter of the lac operon of E. coli, the Streptomyces coelicoloragarase gene dagA promoters, the promoters of the Bacillus licheniformisα-amylase gene (amyL), the promoters of the Bacillus stearothermophilusmaltogenic amylase gene (amyM), the promoters of the Bacillusamyloliquefaciens α-amylase (amyQ), the promoters of the Bacillussubtilis xylA and xylB genes, etc. For transcription in a fungal host,examples of useful promoters are those derived from the gene encoding A.oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. nigerneutral α-amylase, A. niger acid stable α-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A.oryzae triose phosphate isomerase or A. nidulans acetamidase.

The expression vector of the invention may also comprise a suitabletranscription terminator and, in eukaryotes, polyadenylation sequencesoperably connected to the DNA sequence encoding the maltogenicalpha-amylase variant of the invention. Termination and polyadenylationsequences may suitably be derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question. Examples of such sequences arethe origins of replication of plasmids pUC19, pACYC177, pUB110, pE194,pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracycline resistance. Furthermore, the vector may compriseAspergillus selection markers such as amdS, argB, niaD and sC, a markergiving rise to hygromycin resistance, or the selection may beaccomplished by co-transformation, e.g. as described in WO 91/17243.

While intracellular expression may be advantageous in some respects,e.g. when using certain bacteria as host cells, it is generallypreferred that the expression is extracellular. In general, the Bacillusα-amylases mentioned herein comprise a preregion permitting secretion ofthe expressed protease into the culture medium. If desirable, thispreregion may be replaced by a different preregion or signal sequence,conveniently accomplished by substitution of the DNA sequences encodingthe respective preregions.

The procedures used to ligate the DNA construct of the inventionencoding maltogenic alpha-amylase variant, the promoter, terminator andother elements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambroo, et al.,op.cit.).

The cell of the invention, either comprising a DNA construct or anexpression vector of the invention as defined above, is advantageouslyused as a host cell in the recombinant production of a maltogenicalpha-amylase variant of the invention. The cell may be transformed withthe DNA construct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g. by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

The cell of the invention may be a cell of a higher organism such as amammal or an insect, but is preferably a microbial cell, e.g. abacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are gram positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gram negative bacteria such as E.coli. The transformation of the bacteria may, for instance, be effectedby protoplast transformation or by using competent cells in a mannerknown per se.

The yeast organism may favorably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g. Saccharomyces cereviseae. Thefilamentous fungus may advantageously belong to a species ofAspergillus, e.g. Aspergillus oryzae or Aspergillus niger. Fungal cellsmay be transformed by a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known per se. A suitable procedure for transformationof Aspergillus host cells is described in EP 238 023.

In a yet further aspect, the present invention relates to a method ofproducing a maltogenic alpha-amylase variant of the invention, whichmethod comprises cultivating a host cell as described above underconditions conducive to the production of the variant and recovering thevariant from the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in question and obtaining expressionof the maltogenic alpha-amylase variant of the invention. Suitable mediaare available from commercial suppliers or may be prepared according topublished recipes (e.g. as described in catalogues of the American TypeCulture Collection).

The maltogenic alpha-amylase variant secreted from the host cells mayconveniently be recovered from the culture medium by well-knownprocedures, including separating the cells from the medium bycentrifugation or filtration, and precipitating proteinaceous componentsof the medium by means of a salt such as ammonium sulfate, followed bythe use of chromatographic procedures such as ion exchangechromatography, affinity chromatography, or the like.

Screening of Variants with Maltogenic Alpha-Amylase Activity

Variants produced by any of the methods described above may be tested,either prior to or after purification, for maltogenic alpha-amylaseactivity, such as amylolytic activity, in a screening assay whichmeasures the ability of the variant to degrade starch. The screening instep 10 in the above-mentioned random mutagenesis method of theinvention may be conveniently performed by use of a filter assay basedon the following procedure: A microorganism capable of expressing thevariant of interest is incubated on a suitable medium and under suitableconditions for secretion of the variant, the medium being covered withtwo filters comprising a protein-binding filter placed under a secondfilter exhibiting a low protein binding capability. The microorganism isgrown on the second, top filter. Subsequent to the incubation, thebottom protein-binding filter comprising enzymes secreted from themicroorganism is separated from the second filter comprising themicro-organism. The protein-binding filter is then subjected toscreening for the desired enzymatic activity, and the correspondingmicrobial colonies present on the second filter are identified. Thefirst filter used for binding the enzymatic activity may be anyprotein-binding filter, e.g., nylon or nitrocellulose. The second filtercarrying the colonies of the expression organism may be any filter thathas no or low affinity for binding proteins, e.g., cellulose acetate orDurapore™.

Screening consists of treating the first filter to which the secretedprotein is bound with a substrate that allows detection of theamylolytic activity. The enzymatic activity may be detected by a dye,fluorescence, precipitation, pH indicator, IR-absorbance or any otherknown technique for detection of enzymatic activity. The detectingcompound may be immobilized by any immobilizing agent e.g. agarose,agar, gelatine, polyacrylamide, starch, filter paper, cloth; or anycombination of immobilizing agents. For example, amylolytic activity canbe detected by Cibacron Red labeled amylopectin, which is immobilized inagarose. Amylolytic activity on this substrate produces zones on theplate with reduced red color intensity.

To screen for variants with increased stability, the filter with boundmaltogenic alpha-amylase variants can be pretreated prior to thedetection step described above to inactivate variants that do not haveimproved stability relative to the parent CGTase. This inactivation stepmay consist of, but is not limited to, incubation at elevatedtemperatures in the presence of a buffered solution at any pH from pH 2to 12, and/or in a buffer containing another compound known or thoughtto contribute to altered stability e.g., surfactants, EDTA, EGTA, wheatflour components, or any other relevant additives. Filters so treatedfor a specified time are then rinsed briefly in deionized water andplaced on plates for activity detection as described above. Theconditions are chosen such that stabilized variants show increasedenzymatic activity relative to the parent after incubation on thedetection media.

To screen for variants with altered thermostability, filters with boundvariants are incubated in buffer at a given pH (e.g., in the range frompH 2-12) at an elevated temperature (e.g., in the range from 50°-110°C.) for a time period (e.g., from 1-20 minutes) to inactivate nearly allof the parent CGTase, rinsed in water, then placed directly on adetection plate containing immobilized Cibacron Red labeled amylopectinand incubated until activity is detectable. Similarly, pH dependentstability can be screened for by adjusting the pH of the buffer in theabove inactivation step such that the parent CGTase is inactivated,thereby allowing detection of only those variants with increasedstability at the pH in question. To screen for variants with increasedcalcium-dependent stability calcium chelators, such as ethyleneglycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), isadded to the inactivation buffer at a concentration such that the parentCGTase is inactivated under conditions further defined, such as bufferpH, temperature or a specified length of incubation.

The variants of the invention may be suitably tested by assaying thestarch-degrading activity of the variant, for instance by growing hostcells transformed with a DNA sequence encoding a variant on astarch-containing agarose plate and identifying starch-degrading hostcells as described above. Further testing in regard to alteredproperties, including specific activity, substrate specificity, cleavagepattern, thermoactivation, thermostability, pH dependent activity oroptimum, pH dependent stability, temperature dependent activity oroptimum, transglycosylation activity, stability, and any other parameterof interest, may be performed on purified variants in accordance withmethods known in the art as described below.

The maltogenic alpha-amylase activity of variants of the inventiontowards linear maltodextrins and cyclodextrins may be assayed bymeasuring the hydrolysis of maltotriose. Hydrolysis is monitored by theformation of glucose using the GLU-kit (Boehringer Mannheim,Indianapolis Ind.). Hydrolysis of longer maltodextrins, such asmalto-tetraose to -heptaose) and cyclodextrins is monitored by theformation of free reducing ends which is measuredspectrophotometrically.

Alternatively, amylolytic activity can be assayed using the Phadebasmethod (BioRad, Inc., Richmond, Calif.) in which the substrate is awater-insoluble cross-linked starch polymer carrying a blue dye(Phadebas Amylase Test) that is hydrolyzed by amylolytic activity toform water-soluble blue fragments which can then be quantitatedspectrophotometrically.

In cases where the variants of the invention have been altered in thesubstrate binding site, it may be desirable to determine whether suchvariant is capable of performing a transglycosylation reaction, which isdescribed below in Example 1, as is normally observed for CGTases.

Substrate specificity of maltogenic alpha-amylase variants may beassayed by measuring the degree to which such enzymes are capable ofdegrading starch that has been exhaustively treated with theexoglycosylase β-amylase. To screen for variants which show patterns ofdegradation on such a substrate differing from the patterns produced bythe parent CGTase the following assay is performed: β-limit dextrin isprepared by incubating 25 ml 1% amylopectin in Mcllvane buffer (48.5 mMcitrate and 193 mM sodium phosphate pH 5.0) with 24 μg/ml β-amylaseovernight at 30° C. Unhydrolysed amylopectin (i.e., β-limit dextrin) isprecipitated with 1 volume 98% ethanol, washed and redissolved in water.1 ml β-limit dextrin is incubated with 18 μl enzymes (at 2.2 mg/ml) and100 μl 0.2 M citrate-phosphate pH 5.0 for 2 hrs at 30° C. and analysedby HPLC as described above. Total hydrolysis of β-limit dextrin iscarried out in 2M HCl at 95° C. The concentration of reducing ends ismeasured by methods known in the art.

INDUSTRIAL APPLICATIONS

The maltogenic alpha-amylase variants of the invention possess valuableproperties which may be advantageously used in various industrialapplications. In particular, the enzyme finds potential application forretarding or preventing retrogradation, and thus the staling, of starchbased food common in the baking industry.

The variant may be used for the preparation of bread and other breadproducts in accordance with conventional techniques known in the art.

It is believed that the modification of the starch fraction by use ofthe present invention results in increased volume in baked products andimproved organoleptic qualities, such as flavor, mouth feel,palatability, aroma and crust color.

The maltogenic alpha-amylase variant may be used as the only enzyme oras a major enzymatic activity in combination with one or more additionalenzymes, such as xylanase, lipase, glucose oxidase and otheroxidoreductases, or an amylolytic enzyme.

The enzyme variants of the invention also find industrial applicabilityas a component in washing, dishwashing and hard-surface cleaningdetergent compositions. Some variants are particularly useful in aprocess for the manufacture of linear oligosaccharides, or in theproduction of sweeteners and ethanol from starch, and/or for textiledesizing. Conditions for conventional starch conversion processes,including starch liquefaction and/or saccharification processes, aredescribed in, e.g., U.S. Pat. No. 3,912,590 and in EP patentpublications Nos. 252,730 and 63,909.

The variants of the invention also find application in processes for themanufacture of cyclodextrins for various industrial applications,particularly in the food, cosmetic, chemical, agrochemical andpharmaceutical industries.

Therefore in another aspect the invention provides maltogenicalpha-amylase variants for use in a process for the manufacture ofcyclodextrins, in particular α-, β-, δ-, ε-, and/or ζ-cyclodextrins. Ina more preferred embodiment, the invention provides maltogenicalpha-amylase variants for use in a process for the manufacture of α-,β- and γ-cyclodextrins, or mixtures hereof.

In yet another preferred embodiment, the variants of the invention maybe used for in situ generation of cyclodextrins. In this way thevariants of the invention may be added to a substrate containing mediumin which the enzyme variants are capable of forming the desiredcyclodextrins. This application is particularly well suited for use inmethods of producing baked products as described above, in methods forstabilizing chemical products during their manufacture, and in detergentcompositions.

Cyclodextrins have an inclusion ability useful for stabilization,solubilization, etc. Thus cyclodextrins can make oxidizing andphotolytic substances stable, volatile substances non-volatile,poorly-soluble substances soluble, and odoriferous substances odorless,etc. and thus are useful to encapsulate perfumes, vitamins, dyes,pharmaceuticals, pesticides and fungicides. Cyclodextrins are alsocapable of binding lipophilic substances such as cholesterol, to removethem from egg yolk, butter, etc.

Cyclodextrins also find utilization in products and processes relatingto plastics and rubber, where they have been used for different purposesin plastic laminates, films, membranes, etc. Also, cyclodextrins havebeen used for the manufacture of biodegradable plastics.

EXAMPLES

The invention is further illustrated with reference to the followingexamples which are not intended to be in any way limiting to the scopeof the invention as claimed.

Example 1 Construction of Variants of Thermoanaerobacter CGTase withAltered Substrate Specificity

This example describes the construction of CGTase variants with modifiedsubstrate specificity. The variants are derived from a parentThermoanaerobacter sp. CGTase (i.e. the wild type), obtained asdescribed in WO 89/03421 and WO 96/33267.

Bacterial Strains, Plasmids and Growth Conditions

Escherichia coli ME32 was used for recombinant DNA manipulations. Thevariants were expressed in SHA273, a derivative of Bacillus subtilis 168which is apr⁻, npr⁻, amyE⁻, amyR2⁻and prepared by methods known in theart. pCA31-wt is a E. coli-B. subtilus shuttle vector harboring theparent Thermoanaerobacter CGTase, shown in FIG. 1.

DNA Manipulations

DNA manipulations and transformation of E. coli were essentially asdescribed in Sambrook, J., Fritsch, E. J., and Maniatis, T. (1989)Molecular cloning: a laboratory manual, Cold Spring Harbor LaboratoryPress, New York. B. subtilis was transformed using methods known in theart.

Site-Directed Mutagenesis

Mutant CGTase genes were constructed via SOE-PCR method (Nelson andLong, op.cit.) using the Pwo DNA polymerase (Boehringer Mannheim,Indianapolis, Ind.). The primary PCR reactions were carried out with themutagenesis primers 1 and 2 (SEQ ID NO: 3 and 4) plus an upstream or adownstream primer (SEQ ID NO: 5 or 6) on the template strand,respectively. The reaction products were subsequently used as templatein a second PCR reaction together with the upstream and downstreamprimers. The product of the last reaction was digested with StyI andSpeI, and exchanged with the corresponding fragment (640 bp) from thevector pCA31-wt or pCA31-Δ(67-94)(T-CGTase+L82H+D84*+T84bG+F84cY+G84d*+G85*+S86*). The resulting variantplasmids were transformed into E. coli ME32 and vector DNA was purifiedfrom E. coli colonies using the DNA-purification kit from QIAGEN(Qiagen, Inc. Germany). The mutant vectors were finally transformed intoB. subtilis SHA273 for enzyme expression.

The degeneration of mutagenesis primer 2 (SEQ ID NO: 4) containing A orC/G gave rise to two different amino acid sequences. Thus, two variantsof a parent CGTase were constructed. Successful mutations resulted inrestriction sites (Sac II) at positions 7-12 of SEQ ID NO: 3 andpositions 2-7 of SEQ ID NO: 4, which allowed quick screening oftransformants. Mutations were verified by standard DNA sequencingtechniques. The correctness of the StyI-SpeI fragment obtained by PCRwas also confirmed by DNA sequencing.

Production and Purification of CGTase Proteins

Enzymes were produced in transformed SHA273 cells grown in shakeflasksat 30-37° C. in 2*TY media containing 10 μg/l kanamycin. After 68-72 hof growth the culture was pelleted and the supernatant separated fromthe cells by centrifugation. After filtration through a 0.45 μmnitrocellulose filter, the supernatant was directly applied to anα-cyclodextrin-sepharose-6FF affinity column (Monma et al. 1988Biotechnol. Bioeng. 32, 404-407). After washing the column with 10 mMsodium acetate (pH 5.5), the variants were eluted with the same buffersupplemented with 1% (w/v) α-cyclodextrin. Purity and molecular weightof the variants obtained were checked by SDS-PAGE. Proteinconcentrations were determined by measuring the absorption at 280 nmusing a theoretical extinction coefficient at 1.74 ml/mg⁻¹/cm⁻¹.

Enzyme Assays

All assays were performed at pH 6.0 and 37° C. The assay for cyclizationactivity was performed as described by Penning a et al (1995,Biochemistry 34:3368-3376). Starch liquefying activity was measuredusing the Phadebas Amylase Test kit (Pharmacia A/B, Sweden).Transglycosylation activity was assayed in which 2.2 μM of the variantwas incubated with 200 mM maltotriose at 40° C. in 10 mM NaOAc pH 5.0and 1 mM CaCl₂. At different time intervals, aliquots were analyzed byHPLC to measure formation of different maltodextrins. Analyticalseparations of maltodextrins were performed on a Dionex CarboPacPA1-column connected to a Beckman Gold HPLC-system and apulsed-amperometric detector. The gradient was 0-600 mM NaOAc over 15minutes in 0.1 M NaOH. Transglycosylation activity was detected as anincrease in the size of the from three to greater than three glucoseunits covalently linked.

Example 2 Specific PCR Amplification Using Novamyl-Specific PCR Primers

Comparison of Novamyl with CGTases reveals that it is thus far unique inone structural feature: the insertion of a 5 amino acid “loop” in domainA, residues 190 to 194 in the amino acid sequence shown in SEQ ID NO: 1,that affects the enzyme structure near the active site. It is thereforevaluable to have a method of obtaining variants with a similar activesite structure, especially with respect to the Novamyl active site loop.Here we describe such a method using PCR primers specific to the Novamylloop to amplify from natural sources only those clones with this uniquestructural feature.

Step 1. PCR Amplification of Glycosylases with Degenerate Primers

Alignment of amino acid sequences for Novamyl with known CGTases revealsregions of high homology that can be used to design degenerateoligonucleotide primers for use in the PCR amplification of CGTases froma mixed pool of genomic or cDNA. The resulting fragments of a predictedsize range can then be used as template DNA in further PCRamplifications with Novamyl loop-specific primers as described in Step2.

Alignment of 10 amino acid sequences most related to Novamyl was used toidentify two regions of high local homology for the design of degenerateprimers: Primer 1 (SEQ ID NO: 7) corresponding to amino acids 88-93 fromSEQ ID NO: 1 and Primer 2 (SEQ ID NO: 8) corresponding to amino acids417-412 from SEQ ID NO: 1.

Use of these primers on DNA fragments from bacterial sources can, whenused as primers in a PCR reaction under standard conditions, amplify aDNA fragment approximately 1,000 basepairs in length containing thecentral core of related glycosylases. Resulting PCR fragments could thenbe used as templates in step 2, as described below.

Step 2. PCR Amplification Using Novamyl Loop-Specific Primers (FIG. 3)

The following primer pair can be used, corresponding to the degeneratetranslation of the amino acid sequence in the coding (d3, SEQ ID 9) ornoncoding (d4, SEQ ID NO: 10) DNA strand:

Primer d3 (SEQ ID NO: 9): degenerate sense primer corresponding to aminoacids 190-194 from SEQ ID NO: 1.

Primer d4 (SEQ ID NO: 10): degenerate anti-sense primer corresponding toamino acids 194-190 from SEQ ID NO: 1.

Alternatively, it is possible to use the degenerate or exact nucleotidesequence of Novamyl through eight amino acids that contain the Novamylsequence, Phe188-Thr189-Asp190-Pro191-Ala192-Gly193-Phe194-Ser195 (loopunderlined) in both DNA strands as primers in a PCR reaction:

Primer d5 (SEQ ID NO: 11): degenerate sense primer corresponding toamino acids 188-193 from SEQ ID NO: 1.

Primer d6 (SEQ ID NO: 12): degenerate anti-sense primer corresponding toamino acids 195-190 from SEQ ID NO: 1.

Primer 7 (SEQ ID NO: 13): Exact Novamyl sense primer corresponding toamino acids 188-193 from SEQ ID NO: 1.

Primer 8 (SEQ ID NO: 14): Exact Novamyl anti-sense primer correspondingto amino acids 195-190 from SEQ ID NO: 1.

Using the PCR products of step 1 as templates in a PCR reaction togetherwith primer pairs 1 and d4, 2 and d4, 1 and d5, 2 and d6, 1 and 7, or 2and 8, only those DNA sequences encoding the Novamyl loop are expectedto produce DNA fragments of approximately the predicted size (FIG. 1,step 2). Templates lacking the loop-encoding DNA will not produce aproduct under standard PCR conditions with an annealing temperature of58° C.

approximate size of Product Primer pair product A1 1 + d4 321 A2 1 + d6324 A3 1 + 8 324 B1 2 + d3 684 B2 2 + d5 690 B3 2 + 7 690

Step 3. Reconstruction of Full-Length Fragments

Step 2 yields partial coding sequences of glycosylases that contain theNovamyl loop at either the 3′ (A fragments) or 5′ ends (B fragments) ofthe DNA fragments. Reassembly of longer clones containing the loop willrequire the combining of the A and B fragments by SOE-PCR methods knownin the art.

Example 3 Conversion of CGTases into Novamyl-Like Enzymes by RandomRecombination

In this example, the unique active site loop was used to select hybridenzymes with maltogenic alpha-amylase activity from a library of randomrecombinants. In this method, Novamyl and the cyclic maltodextringlucosyl transferase (CGTase) from Bacillus circulans, were randomlyrecombined by the DNA shuffling method of Crameri A, et al., op.cit.Those resulting mutants containing the Novamyl loop were selected usingPCR as described above in Example 2.

Step 1. PCR Amplification and Shuffling of Novamyl and CGTase (FIG. 2)

Specific oligonucleotide primers specific for either the Novamyl codingsequence or the CGTase coding sequenced were designed as shown in SEQ IDNO: 15-20.

The entire Novamyl coding sequence (lacking the signal sequence) wasamplified using the Novamyl-specific primer pair #9 and #10 (SEQ ID NO:15 and 16). Similarly, the mature CGTase coding sequence was amplifiedusing the CGTase-specific primer pair #11 and #12 (SEQ ID NO: 17 and18). Both amplifications were performed using the following reactionconditions: 100 μM each primer, 0.2 mM each of dATP, dCTP, dGTP, andTTP, 2.5 U AmpliTaq polymerase (Perkin Elmer, Inc.), and 1×concentration of the buffer supplied by the manufacturer. PCR wasperformed in a Perkin Elmer Thermocycler, model 2400, with the followingconditions: 5 minutes at 94 C. 25 cycles of 30 seconds at 94 C., 1minute at 58 C, and 2 minutes at 72 C. followed by a final incubation of7 minutes at 72° C.

The resulting PCR products were then subjected to DNA shuffling asdescribed by Stemmer et al. Briefly, the two DNA fragments were mixed inequimolar amounts and randomly digested using DNase I treatment togenerate gene fragments of between 50 and 500 bp. These gene fragmentswere then allowed to anneal to one another and extend in a PCR reactionunder low stringency conditions, resulting in a re-assembling of anintact gene pool containing the reassemble parental DNAs as well aschimeras between the parents. Final amplification of shuffled productswas performed using the general primer pair #13 and #14 (SEQ ID NO: 19and 20) using the PCR conditions described above. Using these primers,all full-length species, both parental and chimeric, were amplified.

Step 2. PCR Amplification Using Novamyl Loop-Specific Primers (FIG. 3)

Those genes within this mixture containing the Novamyl loop were thenselected for as described in Example 2 using the loop-specific primers.In the first round of amplification, the 5′ and 3′ ends of the genescontaining the loop were amplified by using the general primers incombination with the loop specific primers #7 and #8 (SEQ ID NO: 13 and14) to amplify either the 5′ ends of the genes extending to theloop-encoding sequence #13 and #8 (SEQ ID NO: 19 and 14) or the sequenceextending from the loop to the 3′ ends of the genes #7 and #14 (SEQ IDNO: 13 and 20). As described in Example 2, the resulting fragments werethen assembled using SOE PCR to create full-length genes. These productswere then selectively amplified using primer pairs lacking aNovamyl-specific primer to produce only chimeras: #11, #10 and #12 (SEQID NO: 17, 16 and 18) or #10, #9 and #11 (SEQ ID NO: 16, 15 and 17). Inthis way, only those clones that contained either the CGTase 5′ end andthe Novamyl loop or the CGTase 3′ end and the Novamyl loop wereselected.

The final PCR product were then digested with the enzymes Xba I and MluI and inserted into a vector containing an intact signal sequence. Theresulting clones were transformed into Bacillus, and the resultingpolypeptides were sequenced.

3 polypeptides obtained by this method were found to be hybridscontaining an N-terminal sequence from Novamyl and a C-terminal sequencefrom the B. circulans CGTase as follows:

Novamyl amino acids 1-196+CGTase amino acids 198-685

Novamyl amino acids 1-230+CGTase amino acids 232-685

Novamyl amino acids 1-590+CGTase amino acids 596-485.

These results demonstrate that the method is effective for generatingand selecting hybrids containing the

Example 4 Construction of a Variant of Novamyl with CGTase Activity

A variant of Novamyl was constructed that has an altered substratespecificity relative to the parent enzyme, in which the variant has aCGTase-like transglycosylation/cyclization activity not detectable inthe parent enzyme. The variant differs from the parent Novamyl with theamino acid sequence shown in amino acids 1-686 of SEQ ID NO: 1 in thatresidues 191-195 were removed, Phe188 was substituted with Leu andThr189 was substituted with Tyr, termed Δ (191-195)-F188L-T189Y. Thevariant was constructed by sequence overlap extension PCR (SOE PCR)essentially as described by Nelson and Long (op.cit.). SOE PCR consistsof two primary PCRs that produce two overlapping PCR fragments, bothbearing the same modification(s). In a second round of PCR, the twoproducts from the two primary PCRs are mixed without addition oftemplate DNA.

Oligonucleotide Primers Used in the Construction of Δ(191-195)-F188L-T189Y:

Mutagenic Primer 1 (SEQ ID NO: 21) and Primer 2 (SEQ ID NO: 22).Positions 16-21 of SEQ ID NO: 21 and positions 4-9 of SEQ ID NO: 22 arerestriction sites.

The Δ (191-195)-F188L-T189Y were obtained using oligomers A82 (SEQ IDNO: 23) and B346 (SEQ ID NO: 24) as end-primers.

DNA manipulations, transformation of Bacillus subtilis, and purificationof the resulting variant was performed as described above in Example 4.The final purified variant was analysed for the ability to formcyclodextrin from linear starch and compared to the parent Novamylenzyme as described below.

Detection of β-Cyclodextrin

The variant and the parent Novamyl enzyme were diluted with 10 mMcitrate buffer pH 6.0 in order to obtain an equivalent proteinconcentration prior to assay.

The cyclisation reaction mixture in a final volume of 1 ml contained:

0-50 μl enzyme or variant diluted in 10 mM sodium citrate buffer pH 6.0

500 μl 10% (w/v) Paselli SA2 (AVEBE, Foxhol, The Netherlands) dissolvedin 10 mM citrate buffer pH 6.0 for a final solution of 5%

The reaction mixture was pre-incubated in a 50° C. water bath for 10min. before adding the enzyme or variant, and at one-min time intervalsa 100 μl sample was put on ice for further analysis.

β-cyclodextrin was quantitated on the basis of formation of a stablecolourless inclusion complex with phenolphthalein; thus, the colour ofthe solution decreases with as the amount of β-cyclodextrin detectedincreases.

To each of the 100 μl samples from the cyclization reaction 900 μl of aworking solution (3 ml of 3.75 mM phenolphthalein added to 100 ml 0.2 MNa₂CO₃, pH 9.7) was added, and the absorbance immediately read at 552nm. β-cyclodextrin was quantitated on the basis of a calibration curveprepared in a final volume of one-ml as follows:

0-50 μl 2 mM β-cyclodextrin (0-100 nmol)

50-0 μl milli-Q water

900 μl working solution

50 μl 10% Paselli SA2

A new calibration curve was made for each new preparation of Paselli SA2solution.

The results of the cyclization assay are presented in the table below asβ-cyclodextrin formation (mmol/mg enzyme) for the variant Δ(191-195)-F188L-T189Y and for the parent enzyme, Novamyl:

Time (min) Variant Novamyl 0 0 0 2 160 0 3 230 0 4 240 0 5 320 0 6 380 07 390 0 8 500 0 10 680 0

The results clearly demonstrate that the variant, unlike the parentNovamyl enzyme, can form β-cyclodextrin.

Example 5 Construction of a CGTase Variant with Ability to Form LinearOligosaccharides

This example describes the construction of a CGTase variant derived froma parent Thermoanaerobacter CGTase.

Mutant CGTase genes were constructed via SOE-PCR method as described inExample 1. The primary PCR reactions were carried out with themutagenesis primers A91 (SEQ ID NO: 26) and A90 (SEQ ID NO: 25) plus anupstream or a downstream primer (SEQ ID NO 5 or 6) on the templatestrand, respectively. The product of the last reaction was digested withBst1107 I and Pst I, and exchanged with the corresponding fragment (250bp) from the vector pCA31-wt orpCA31-(T-CGTase+F189L+*190D+*191P+*192A+*193G+*194F+D195S). Successfulmutations resulted in restriction sites (Xma I) at positions 4-9 of A91(SEQ ID NO: 26) and positions 11-16 of A90 (SEQ ID NO: 25), whichallowed quick screening of transformants. The following mutations wereverified by standard DNA sequencing techniques:

Y260F+L261G+G262D+T263D+N264P+E265G+V266T+*266aA+*266bN+D267H+268V

*194aT+*194bD+*194cP+*194dA+*194eG+D196S+Y260F+L261G+G262D+T263D+N264P+E265G+V266T+*266aA+*266bN+D267H+P268V

Example 6 Properties of CGTase Variant with Ability to Form LinearOligosaccharides Inhibition of Starch Retrogradation

The first variant prepared in Example 5 was tested for its ability toinhibit starch retrogradation was tested as follows:

730 mg of 50% (w/w) amylopectin slurry in 0.1 M sodium acetate, at aselected pH (3.7, 4.3 or 5.5) was mixed with 20 μl of an enzyme sample,and the mixture was incubated in a sealed ampoule for 1 hour at 40° C.,followed by incubation at 100° C. for 1 hour in order to gelatinize thesamples. The sample was then aged for 7 days at room temperature toallow recrystallization of the amylopectin. A control without enzyme wasincluded.

After aging, DSC was performed on the sample by scanning from 5° C. to95° C. at a constant scan rate of 90° C./hour. The area under the firstendothermic peak in the thermogram was taken to represent the amount ofretrograded amylopectin, and the relative inhibition of retrogradationwas taken as the area reduction (in %) relative to the control withoutenzyme.

The result was a relative inhibition of 21%.

Reaction Pattern with Starch

The variant was compared with Novamyl and with Thermoanaerobacter CGTaseby determining the reaction products formed after 24 hours incubation in5% (w/v) amylopectin using 50 mM sodium acetate, 1 mM CaCl2, pH 5.0 at50° C. The reaction products (in % by weight) were identified andquantitated using HPLC.

Oligosaccharide Novamyl CGTase Variant G10 — — 0.7 G9 — — 1.5 G8 — — 2.4G7 — — 1.9 G6/α-CD — 53.9 23.1  G5 — — 6.1 G4 — — 8.1 G3/γ-CD — 12.014.5  G2 97.9 — 11.5  G1  2.1 — 6.8 β-CD 34.1 23.1 

The results show clearly that whereas the parent CGTase exclusivelyforms cyclodextrins, the reaction pattern of the variant has beenchanged to form both cyclodextrins and linear maltodextrins as initialproducts.

1. A method for producing a variant of a parent cyclodextringlucanotransferase, comprising modifying the amino acid sequence of aparent cyclodextrin glucanotransferase by substituting, inserting ordeleting one or more amino acids of said amino acid sequence, whereinsaid substitution is a substitution an amino acid residue which ispresent in a corresponding position in the amino acid sequence of aminoacids 1 to 686 of SEQ ID NO:1 but which is not present in the amino acidsequence of the parent cyclodextrin glucanotransferase; wherein saidinsertion is an insertion of an amino acid residue which is present in acorresponding position in the amino acid sequence of amino acids 1 to686 of SEQ ID NO:1 but which is not present in the amino acid sequenceof the parent cyclodextrin glucanotransferase; and wherein said deletionis a deletion of an amino acid residue which is present in the parentcyclodextrin glucanotransferase but which is not present in the aminoacid sequence of amino acids 1 to 686 of SEQ ID NO:1, and wherein thecyclodextrin glucanotransferase variant forms linear oligosaccharideswhen acting on starch.
 2. The method of claim 1, wherein the parentcyclodextrin glucanotransferase is from a strain of Bacillus.
 3. Themethod of claim 1, wherein the parent cyclodextrin glucanotransferase isfrom a strain of Brevibacterium.
 4. The method of claim 1, wherein theparent cyclodextrin glucanotransferase variant is from a strain ofClostridium.
 5. The method of claim 1, wherein the parent cyclodextringlucanotransferase variant is from a strain of Corynebacterium.
 6. Themethod of claim 1, wherein the parent cyclodextrin glucanotransferasevariant is from a strain of Klebsiella.
 7. The method of claim 1,wherein the parent cyclodextrin glucanotransferase variant is from astrain of Micrococcus.
 8. The method of claim 1, wherein the parentcyclodextrin glucanotransferase variant is from a strain ofThermoanaerobacter
 9. The method of claim 1, wherein the parentcyclodextrin glucanotransferase variant is from a strain ofThermoanaerobacterium.
 10. A cyclodextrin glucanotransferase variantprepared by the method of claim
 1. 11. A method for producing a variantof a parent cyclodextrin glucanotransferase, comprising (a) modifyingthe amino acid sequence of a parent cyclodextrin glucanotransferase bysubstituting, inserting or deleting one or more amino acids of saidamino acid sequence, wherein said substitution is a substitution anamino acid residue which is present in a corresponding position in theamino acid sequence of amino acids 1 to 686 of SEQ ID NO:1 but which isnot present in the amino acid sequence of the parent cyclodextringlucanotransferase; wherein said insertion is an insertion of an aminoacid residue which is present in a corresponding position in the aminoacid sequence of amino acids 1 to 686 of SEQ ID NO:1 but which is notpresent in the amino acid sequence of the parent cyclodextringlucanotransferase; and wherein said deletion is a deletion of an aminoacid residue which is present in the parent cyclodextringlucanotransferase but which is not present in the amino acid sequenceof amino acids 1 to 686 of SEQ ID NO:1, (b) testing the variantcyclodextrin glucanotransferase for the ability to form linearoligosaccharides when acting on starch; (c) producing the variantcyclodextrin glucanotransferase by cultivating a host cell comprising anucleic acid sequence encoding the variant cyclodextringlucanotransferase; and (d) recovering the cyclodextringlucanotransferase variant.
 12. A method for producing a variant of aparent cyclodextrin glucanotransferase, comprising: (a) cultivating ahost cell comprising a nucleic acid sequence encoding a variant of acyclodextrin glucanotransferase, wherein said cyclodextringlucanotransferase variant comprises and insertion, substitution ordeletion of one or more amino acids, wherein said substitution is asubstitution an amino acid residue which is present in a correspondingposition in the amino acid sequence of amino acids 1 to 686 of SEQ IDNO:1 but which is not present in the amino acid sequence of the parentcyclodextrin glucanotransferase; wherein said insertion is an insertionof an amino acid residue which is present in a corresponding position inthe amino acid sequence of amino acids 1 to 686 of SEQ ID NO:1 but whichis not present in the amino acid sequence of the parent cyclodextringlucanotransferase; and wherein said deletion is a deletion of an aminoacid residue which is present in the parent cyclodextringlucanotransferase but which is not present in the amino acid sequenceof amino acids 1 to 686 of SEQ ID NO:1, and wherein the cyclodextringlucanotransferase variant forms linear oligosaccharides when acting onstarch. (b) recovering the cyclodextrin glucanotransferase variant. 13.The method of claim 12, wherein the cyclodextrin glucanotransferase isderived from a strain of Bacillus, Brevibacterium, Clostridium,Corynebacterium, Klebsiella, Micrococcus, Thermoanaerobacter orThermoanaerobacterium.
 14. A cyclodextrin glucanotransferase variantprepared by the method of claim
 12. 15. A method for producing a variantof a parent cyclodextrin glucanotransferase, comprising: (a) cultivatinga host cell comprising a nucleic acid sequence encoding a variant of acyclodextrin glucanotransferase, wherein said cyclodextringlucanotransferase variant comprises and insertion, substitution ordeletion of one or more amino acids, wherein said substitution is asubstitution an amino acid residue which is present in a correspondingposition in the amino acid sequence of amino acids 1 to 686 of SEQ IDNO:1 but which is not present in the amino acid sequence of the parentcyclodextrin glucanotransferase; wherein said insertion is an insertionof an amino acid residue which is present in a corresponding position inthe amino acid sequence of amino acids 1 to 686 of SEQ ID NO:1 but whichis not present in the amino acid sequence of the parent cyclodextringlucanotransferase; and wherein said deletion is a deletion of an aminoacid residue which is present in the parent cyclodextringlucanotransferase but which is not present in the amino acid sequenceof amino acids 1 to 686 of SEQ ID NO:1; (b) transforming a host cellwith the nucleic acid sequence encoding the variant; (c) cultivating thetransformed host cell to express the variant; (d) testing the variantcyclodextrin glucanotransferase for the ability to form linearoligosaccharides when acting on starch; (e) producing the variantcyclodextrin glucanotransferase by cultivating a host cell comprising anucleic acid sequence encoding the variant cyclodextringlucanotransferase; (f) recovering the cyclodextrin glucanotransferasevariant.